DE1065133B - Process for cleaning raw parotene obtained from saliva or salivary gland - Google Patents

Description

The invention relates to the purification of the raw active ingredient from saliva and salivary glands and parotene in particular a process for obtaining these substances in pure crystalline form.

The raw active ingredient used here, i.e. raw parotene, is a raw protein that is extracted from the parotid gland, in particular from cattle, isolated on the basis of the "Salivary gland endocrine theory" derived from T. Ogata will.

This raw parotene can be obtained in the following ways:

The u of fat tissue. Like. Liberated and crushed in a meat grinder fresh bovine parotid gland are treated with the 6-fold amount of water, the mixture with Sn-NaOH adjusted to p _H, 8.0 to 8.2, with a small amount of toluene added and extracted with stirring for 3 hours and this extraction repeated. The (liquid) extract is cloudy and dark red in color. The two extracts are combined, their p _H with 5N HCl to 5.4, allowed to stand overnight in a cold room and then centrifuged ao. The centrifuged precipitate is stirred with the 3- to 4-fold amount of water to form a paste, which was reacted with Sn-NaOH to a p _H 8.0 to 8.2, whereupon it at - refrozen, 3 cc of 15 ° C to 4 became. The frozen material is then allowed to thaw at 18 ° C. on a porcelain filter plate covered with a cotton cloth. The nitrated or centrifuged enamel solution is red but not clear. The colored solution is adjusted with 5N HCl to p _H 5.4 and allowed to stand overnight on ice. The precipitate formed is filtered, washed three times with 20-fold amount of cold acetone washed containing 0.05 ° / ₀ HCl, and suction filtered. After the decolorization, it is further washed with a suitable amount of acetone. The precipitate is then neutralized with aqueous ammonia while stirring, washed twice with about 15 times the amount of absolute acetone and _{dried over concentrated H 2} SO ₄ in vacuo. In this way, 0.8 ° / ₀ of the starting material to "Rohparotin" are obtained.

The parotene promotes the calcification of the teeth in a remarkable way and reduces the calcium level in the serum. In his book "Human Biochemistry", 3rd edition (1951), Kleiner stated that im Saliva contains globulin, but it only speaks indefinitely of a globulin group. That However, parotin is a newly discovered protein that belongs to the globulin group and is also used in treatment of arthritis and other similar diseases of the joints and bone tissue is effective. These Substance is not identical to any of the substances described by Kleiner.

The purified parotin is a product of high therapeutic value that is used in the physiological we-process for cleaning

from Speidiel or salivary glands

obtained raw parotene

Applicant:
Teikoku hormones Mfg. Co., Tokyo

Representative: Dipl.-Ing. H. Leinweber, patent attorney,
Munich 2, Rosental 7:

Claimed priority:
Japan August 18 and August 19, 1943

Tomosaburo Ogata, Akira Ogata, Yosoji Ito, jTokio,
and Akira Mizutani, Nagoya (Japan),
have been named as inventors i

kung is much stronger than the protein-like obtained from saliva or salivary glands by known processes Extracts that are non-toxic, readily soluble in water and suitable for injection

This, according to already known methods, consists of saliva The raw parotene available from the salivary glands is rather impure and has a relatively weak physiological effect.

To get from such extracts of saliva or salivary glands To obtain a purer product, various cleaning methods have been studied in detail], to obtain an active ingredient in crystalline form.

According to the invention, a preparation can now be produced, its physiological effectiveness ten times is stronger than that of the preparations obtained so far. It was also found that this preparation; clinical interesting and effective in the treatment of arthritis deformans and chondrodystrophia fetalis, etc. is so that it offers very good prospects in terms of therapeutic use.

According to the invention obtainable from the saliva or salivary glands in the above manner crude active substance is extracted with an aqueous weakly alkaline medium from a p _H 8.0, and subjected the extract to fractional precipitation with different strong ammonium sulphate solutions or sodium sulfate and then ammonium sulfate solutions to the active substance from the separate inactive substance. It can be repeated in this way until the active substance is deposited in crystalline form, whereupon the same

909 627/385

3 4

if necessary repeatedly water-dried in weakly alkaline. The product thus obtained was purified

Dissolved medium, the solution is still dialyzed and parotene. The yield was about 0.7 g, that is

the dialysate can be freeze-dried. 3 to 4 ° / ₀ of the Rohparotins.

Regarding the physicochemical and chemical

Properties and the biochemical effect of 5 Example 2
Purified product is added to the water or physiological saline solution published in "Endocrinologia - To the raw active ingredient", Vol. I, No. 1, pp. 1 to 50 (1954), work by Yosoji Ito "Biochemical studies in the salivary p _H with n-NaOH solution (using gland hormone," in particular pp 14 to 26 and 41 to 47, cresol or phenol red as indicator) to 8.0 einverwiesen. ίο put and the mixture to dissolve overnight

The crystalline preparation produced according to the invention was left to stand. This solution is physiologically very strong when stirred, is non-toxic, nf> water & the saturated ammonium sulphate solution easily soluble drop by drop, therefore suitable for injection and for, * give until the concentration is very valuable (while maintaining therapeutic purposes. *** a p _H of 8.0) 7% achieved.

In the following examples, execution 15 if the solution has stood for a period of time

forms of the invention described. has, the inactive precipitate is separated and

.3. · “·. ■■ j thereby obtain a clear solution of the active ingredient.

'' P ¹⁶ From the inactive precipitation which still has certain quantities

To 20 g of the Rohparotins was 1 1 a 0.9 ° / ₀ contains sodium-active substances, further amounts of active substance NaCl solution may optionally, 2% butanol were whereupon obtained in the same manner 20 by the Ph of the solution, with 5N NaOH adjusted to 8.0 this precipitate was dissolved in a weakly alkaline solution in order to dissolve the parotene in it. The solution and to this solution or to the dialysate the solution was stirred overnight at 0 to - 5 ⁰ C allowed to stand, so that while maintaining the p _H 8.0 Ammoniumsulfatsich the parotin could dissolve completely. Add to this solution until the concentration of the ammonium solution has reached a saturated sulfate 7% dropwise with stirring. If the precipitated inactive (NH ₄ ) ₂ SO ₄ solution is added until the concentration of substance is separated off, the solution with (NH ₄ ) ₂ SO _{4 was} 7%, whereupon the solution of the active substance overnight. This solution can now be left to stand in a cold room before it has been filtered, the main part obtained as described above. The filtrate was clear and yellow in color. be united.

The solution was brought to p _H 5.4 with n-HCl 3 °, the solution of the active substance in 7 ° / _o sodium ammonium

and left to stand overnight in a cold room, before the monium sulfate solution is now countered with n-HCl solution

it was centrifuged. The precipitate formed was adjusted to bromocresol green as an indicator at 4.8 to 5.0,

in 3 to 4 times the amount of distilled water at Man, this solution is then left to stand overnight, whereupon

P ₁₁ 8.0 dissolved and this solution is separated dropwise under the precipitated active substance.

Stirring with a saturated (NH _{4) 2} SO ₄ solution VCR 35 for this precipitation is the same or 1 ^ ^1-fold

continues until the concentration of (NH _{4) 2} SO ₄ 25 ° where / ₀ off volume of water and the p _H (with Cresol

made (p _H 6.8). This solution was again brought to 8.0 via or phenol red), causing the precipitation

Left to stand overnight in the cold room and then go into solution (any insoluble substance present

centrifuged. 2 g of precipitation were obtained. is discarded). After adding saturated am-

Four times as much distilled monium sulfate solution was added to this precipitation up to a concentration of water. After the precipitation at p _u 8.0 in ammonium sulfate of 25 ° / ₀ (you can also go to the con solution, a saturated (NH ₄ ) ₂ SO ₄ concentration of the ammonium sulfate was added dropwise by adding the seeded solution until the concentration-saturated ammonium sulfate solutions to the original akan (NH ₄ J ₂ SO ₄ 20 ° / ₀ was (p _H 6.8 to 7.0). There was tive 7 ° / _o aqueous solution of ammonium sulfate to _25/0 boost °), a crystalline precipitate, the solution is left to stand in the cold and left to stand overnight and then centrifuged.

The precipitate obtained in this way weighed 1.2 g, which is about 6% of the value. Then it becomes the same again or

Corresponding to raw parotene. given lVj times the volume of water and the p _u

• For this precipitate, 4 times the amount of distil- was adjusted to 8.0 in order to dissolve it (any existing

of water required was added and the p _H of the solution with 50 insoluble material is discarded). To this solution

n-NaOH adjusted to 8.0, in order to add the precipitate or to the dialysate of which, at p _n 8.0, saturated am-

to solve. Then, a monium sulfate solution was added dropwise with stirring to adjust the concentration

Saturated (NH ₄ ) ₂ SO ₄ solution added until the on it _{to bring to 20 ° / 0} , whereupon the solution stand-

^: Concentration of (N Hj) ₂ SO _{4 was} 15 ° / ₀ . The solution is left until the active precipitates and then

was left to stand overnight in the ice room and 55 can be separated.

• then centrifuged. To the precipitate, the This treatment sequence was the precipitation is optionally further 3-fold amount of water and the p _H then repeated twice, whereby only the concentration of n-NAOE H adjusted to 8.0, so that the precipitation of the ammonium sulfate from 20 to 15 ° / ₀ and then ■ resolved. The solution was in running water with finally changing to 12.5 ° / ₀ .
Using a cellophane membrane dialyzed to the 60 marked with the 15 ° / _o aqueous solution of ammonium sulfate conservation (NH ₄ J ₂ SO ₄ to remove. By addition of n-HCl to tene precipitation is crystalline in the main, with the p _H 5 , 4 Precipitation took place, which was centrifuged overnight in a cold room after the 12.5% ammonium sulphate solution was left to stand. The prey consists of practically pure crystals of the active precipitate, twice the amount of distilled substance.

Water was added and the p _H to 8.0 then 65 The active precipitate thus obtained is reacted with n-NaOH,

set, whereby, yourself. the precipitate dissolved. if necessary, several times with water at p _H 4.8

This solution was washed again using one to 5.0, in a small amount weak

Cellophane membrane dialyzed in distilled water, dissolved in alkaline solution and dialyzed, whereupon the

'namely 2 to 3 days, was until the p _H, 6.6 to 6.8. clear solution dialyzed in the cold and dried The dialyzed solution was transferred to a cooling device 70 by setting the dialyzed solution to p _H 4.8

Claims (1)

to 5.0 obtained precipitate dehydrated with acetone. This precipitate is again in a weak or this precipitate is again dissolved in a slightly alkaline solution and dialyzed. To the Dissolved alkaline solution, dialyzed and the dialyzed solution is dropwise the saturated ized solution is dried in the cold. Ammonium sulphate solution added at _pH 7.0 until the In the method described, if necessary, the concentration is 7 ° / ₀ . After deposition of the manoeuvrable a small amount appears, an antiseptic, inactive precipitate is at _pH 6.8 more ammonium z. B. toluene or butanol can be used. Monium sulfate solution added until the concentration In the following, the yields of active substances are 1½ ° / ₀ . Now the active ingredient falls in the form of indicated, the crystals from the process described. can be obtained (the percentages relate to the i ₀ This fraction can, as described in Example 1, Weight of the raw material used as starting material): by fractional precipitation with 12.0 to 12.5% a) 25 "/ ° / ₀ f ^^^ S ^ T be further purified _cent, ammonium sulfate precipitation 20th The end- b) 20% ammonium sulphate precipitates 10 · /. ^{Prey: 240 m} S (° ' ⁸ ° / « ^des Rohparotins). c) 15% ammonium sulfate precipitation (Crystals); ····:; ·; ·· ^{about 5} ^ ' ⁵ claims: d) 12.5 ⁰ J ₀ IgC ammonium sulphate precipitation (Crystals) about 1 ° / ₀ ,.,. , ". . _. ,,, ^v '1. Method of purifying from saliva or These figures were obtained in experiments, the salivary glands in weakly alkaline p _H by were carried out during the winter. Extraction and felling in the weakly acidic range After the purity determination by electrophoresis obtained raw parotene using (using the Tiselius apparatus) possesses the ammonium sulfate or sodium sulfate as in the protein by precipitation with logger ammonium sulfate solution chemistry known per se precipitation salts, thereby fraction obtained has a purity of 80 to 85 ° / _0, which comprises reacting the aqueous at _pH 8.0 in 12.5 ° / _o obtained strength ammonium sulfate solution 35 parliamentary groups medium dissolved Rohparotin by first adding tion such from 90 to 95 ° / ,. a saturated ammonium sulfate solution up to one If a rabbit has about 1 mg of the crystalline ammonium sulfate concentration in the solution of 7 ° / ₀ Preparation is injected per 2 kg body weight, freed from a part of the contamination from the the calcium levels of the blood serum to 15 _^0/0 overall filtrate is the active ingredient by acidification to p _H 4.8 lowers. This shows that the crystalline fraction _3o deposited to 5.4, this again at _pH 8 in water is ten times more effective than the raw active ingredient. When dissolved, and at a 25 ° / _o by weight concentration of ammonium it is injected into humans, no monosulfate precipitates occur, this process occurs at a 20- and unpleasant side reactions. i5 ° l ^ gene and, optionally, 12.5 ° / ₀ sodium Ammon Crystal parotene is a protein that almost does not repeat sulfate concentration, the precipitate contains ash; isoelectric point p _H 5.7 (antimony turn _{35 H} 8 solves at p in water, the solution counter electrode); etj? = - 85.7 ° (10 cm, 0.618 g / 100 cc dialyzed in water, the dialysate to p _H 4.8 to 5.4 H ₂ O). Its absorption spectrum (brings at _pH 7.0 in aqueous, the precipitate obtained is optionally solution) shows a running indefinitely absorption again at _pH 8.0 and dissolved again against water until tion curve at a ηιμ under 235 lying shaft to p _H 6.6 to 6.8 dialyzed and the dialysate freeze lengths; the first maximum occurs at 277 ± 0.5 πιμ and ₄₀ dries or the acid from the dialysates, the second maximum occurs at 405 πιμ. dehydrated products filled with acetone. The protein is relatively unstable, 2. Modification of the procedure for purification of easily decomposed and disintegrated by heat or acid. Raw parotene according to claim 1, characterized in that even if it is referred to as moisture for a long time, that can be done in an aqueous medium running comes. 45 p _H 8.0 dissolved raw parotene instead of ammonia Example 3 sulfate at a 7 ° / _o solution of sodium sulfate concentration pre-cleaned, the filtrate on a sodium sulfate To 30 g of the crude active substance is in terms of volume concentration of 17.7 ° / ₀ brings the precipitate dissolves 50 times as much water or physiological table salt in a weakly alkaline medium, dialysed, at solution given; after the dissolution at setting of 50 p _H 7.0, the solution by addition of sodium sulfate p _H of the solution to 8.0 (using n-NaOH solution indi- to a concentration of sodium sulfate in the er: cresol or phenol red) is from 7% brings a 20 ° / ₀ solution, after separation of the low Sodium sulfate solution with stirring dropwise to the filtrate to-beat again at _pH 7 sodium sulfate given until the concentration reaches 7%. After / ₀ adds up to a concentration of 15 °, the If left to stand for some time, the inactive precipitate produced becomes a weakly alkaline precipitate blow carried away. Medium dissolves, dialyzed, the dialyzate at _pH 7 with Then, further sodium sulfate to ammonium sulphate to a concentration of 7 ° / ₀ Filtrate, in which the active substance is dissolved, brings ammonium sulfate, separates the precipitate, which p _H where 6,6 until the concentration of 17.7 ° / ₀ is. Filtrate to an ammonium sulfate concentration of 15 ° / ₀ After some standing the precipitated active is 60 applies, the precipitate dissolves at _pH 8 in the water, fabric separated. dialyzed and further processed according to claim 1. This precipitation is in a weakly alkaline 3. Process according to Claims 1 and 2, thereby Solution dissolved and dialyzed, whereupon the p _H denotes that NaOH is used for alkalization dialyzed solution is adjusted to 7.0 and sodium sulfate is applied. is added until its concentration reaches 7%. 65 4. The method according to claim 1, characterized After a certain amount of inactive has been separated off, it is noted that HCl or CH ₉ COOH for acidification Precipitate in turn sodium sulfate is used in p _H 7.0,
added to the Na ₂ SO ₄ concentration to 15 ° / ₀ bring; the resulting active precipitation will be Considered publications: severed. 70 German patent specifications No. 706 578, 719 026. © 909 627/385 9.59